The genome and proteome of the Kluyvera bacteriophage Kvp1 – another member of the T7-like Autographivirinae
© Lingohr et al. 2008
Received: 18 September 2008
Accepted: 20 October 2008
Published: 20 October 2008
Kluyvera, a genus within the family Enterobacteriaceae, is an infrequent cause of human infections. Bacteriophage Kvp1, the only bacteriophage isolated for one of its species, Kluyvera cryocrescens, is a member of the viral family Podoviridae.
The genome of Kvp1, the first Kluyvera cryocrescens-specific bacteriophage, was sequenced using pyrosequencing (454 technology) at the McGill University and Genome Québec Innovation Centre. The two contigs were closed using PCR and the sequence of the terminal repeats completed by primer walking off the phage DNA. The phage structural proteome was investigated by SDS-PAGE and mass spectrometry.
At 39,472 bp, the annotated genome revealed a closer relationship to coliphage T3 than T7 with Kvp1 containing homologs to T3 early proteins S-adenosyl-L-methionine hydrolase (0.3) and protein kinase (0.7). The quantitative nature of the relationships between Kvp1 and the other members of the T7-like virus genus (T7, T3, φA1122, φYeO3-12, Berlin, K1F, VP4 and gh-1) was confirmed using CoreGenes.
matrix-assisted laser desorption ionization
- QqTOF MS:
quadrupole time-of-flight mass spectrometry
tandem mass spectrometry.
The T7-like bacterial viruses are members of the Podoviridae – phages with short tails – and are characterized by a simple but elegant temporal transcriptional control system . The early genes are transcribed by the host RNA polymerase while the middle and late regions are transcribed by a single subunit phage-encoded RNA polymerase which recognizes unique 23 bp promoters sequences . These viruses are one of the most common types of bacteriophages with 26–29 defined or tentative species according to the VIII report of the International Committee on the Taxonomy of Viruses [3, 4]. Most of the host species are members of the γ-Proteobacteria (Erwinia, Escherichia, Klebsiella, Morganella, Pseudomonas, Salmonella, Vibrio, and Yersinia) but viral isolates also infecting α-Proteobacteria (Caulobacter, and Rhizobium) have been isolated. Fifteen T7-like phages have been sequenced and deposited with GenBank. As a result of a reanalysis, at the protein level, of relationships within the "T7-like viruses" this group of bacteriophages have been classified into the subfamily Autographivirinae which currently possesses three genera: T7-like, Sp6-like and φKMV-like viruses . Kvp1, the first Kluyvera cryocrescens-specific bacteriophage, was isolated from the Matanza River in Buenos Aires (Argentina) by Gadaleta and Zorzopulos . Morphologically this phage is a member of the Podoviridae. Eleven clones derived from AluI or HaeIII digestion of the viral DNA were sequenced, by these authors, revealing strong sequence similarity to coliphage T7. To further analyze the correct taxonomic position of this virus we have completed the sequence of its genome noting its very close similarity to Yersinia phage Berlin and coliphage T3.
Results and discussion
Pyrosequencing (454 technology) has been used to determine the sequence of the genomes of Bacillus thuringiensis phage 0305φ8-36  and coliphage JK98 , and, in this incidence, the genome of Kvp1. Sequencing resulted in 2 contigs with 53-fold coverage. While this type of sequencing can result in potential errors at oligonucleotide runs, none were observed in the data on Kvp1. The gap, representing 0 bp, was closed by PCR amplification and ABI sequencing; while the nature of the termini were verified by primer walking off phage DNA template. The total genome is 39,472 bp with 194 bp terminal direct repeats, and a base composition of 48.6 mol%G+C – characteristics remarkably consistent with other T7-like phages. By comparison, the genomes of T7-like phages range from 37.4 kb (Pseudomonas phage gh-1) to 45.4 kb (Erwinia phage Era103) while the reported terminal repeats range from Yersinia phage φA1122 at 148 bp to Pseudomonas aeruginosa phage LKD16 at 428 bp.
No tRNA genes were discovered, which was not an unexpected observation since no T7-like phages have been found to harbour them; 46 ORFs were delineated encoding protein products which show the strongest sequence similarity to gene products (Gps) from Yersinia phage Berlin (NC_008694). To investigate the relationships further we employed two homology tools, one of which function at the DNA sequence level (Mauve) and one, CoreGenes, which compares proteins.
Our data conclusively demonstrate that Kluyvera virus Kvp1 is a member of T7-like virus genus of the Podoviridae subfamily Autographivirinae. It differs from phages such as T3 and φYeO3-12 which exhibit capsid frameshifting at lysyl residues, by ribosomal slippage at polyU residues (phenylalanine) – a property it shares with Yersinia phage φA1122.
Materials and methods
Purification of phage wV8
Bacteriophage Kvp1 (HER400) and its host K. cryocrescens strain HER1400 were received from the Felix d'Hérelle Reference Center for Bacterial Viruses at Université Laval (Québec, QC, Canada). The phage was propagated at 30°C using standard protocols, precipitated using polyethylene glycol 8000 and purified through two rounds of CsCl equilibrium gradient centrifugation .
The DNA was isolated using the SDS-proteinase K protocol of Sambrook and Russell (2001) and was submitted to the McGill University and Génome Québec Innovation Centre (Montréal, QC, Canada) for DNA sequencing. This resulted in two contigs which were closed using PCR with custom primers and, standard dideoxy sequencing of the amplicon (University of Guelph, Laboratory Services, Guelph, ON, Canada). The termini were determined by primer walking.
The genome was screened for tRNA-encoding genes using Aragorn  and tRNAScan ; and, for protein encoding genes using Kodon (Applied Maths, Austin, TX) and PSI-BLAST . Rho-independent terminators identified using TransTerm  at http://nbc11.biologie.uni-kl.de/framed/left/menu/auto/right/clusterinfo2. Phage-specific promoters were discovered using PHIRE  and displayed using WebLogo . The sequence of this bacteriophage has been deposited with GenBank (accession no. FJ194439).
Whole genome comparisons
SDS-PAGE  was carried out on CsCl-purified phage particles along with the PageRuler Unstained Protein Ladder (Fermentas, Burlington, ON, Canada) stained with Coomassie brilliant blue R250 and characterized using Bionumerics software (Applied Maths). Bands were further characterized by in situ trypsin digestion and mass spectrometry. Briefly, the excised gel bands were destained until colorless, and dried using a SpeedVac. Following reduction with DTT and alkylation with iodoacetamide, the protein was digested with 10 ng of sequencing grade trypsin (Calbiochem) in 25 mM NH4HCO3 (pH 7.6) at 37°C overnight. The proteolytic peptides were extracted, and cleaned up by a C18 Ziptip (Millipore). MALDI data were acquired using an Applied Biosystems/MDS Sciex QStar XL quadrupole time-of-flight (QqTOF) mass spectrometer under a nitrogen laser (337 nm), and 2,5-dihydroxybenzoic acid was used as the matrix. All peptide fingerprinting masses (m/z) on the MS spectrum were compared with the theoretical values generated in-silico by MS-Digest, a ProteinProspector program developed in the UCSF Mass Spectrometry Facility http://prospector.ucsf.edu/. The individual peptide sequence was identified by MALDI MS/MS measurements on the same instrument using argon as the collision gas.
A.K. is supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. We thank Rob Lavigne for his critical review of the MS. P-J.C. holds a predoctoral fellowship from the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (I.W.T., Belgium).
- Molineux IJ: The T7 Group. The Bacteriophages (Edited by: Calendar R). New York, NY: Oxford University Press 2006, 277–301.
- Chen Z, Schneider TD: Information theory based T7-like promoter models: classification of bacteriophages and differential evolution of promoters and their polymerases. Nucleic Acids Research 2005, 33:6172–6187.PubMedView Article
- Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball A: Virus Taxonomy. VIIIth Report of the International Committee on Taxonomy of Viruses (Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball A). New York, NY: Elsevier Academic Press 2005, 35–85.
- Hendrix RW, Casjens SR: Myoviridae, Siphoviridae, Podoviridae. Virus Taxonomy. VIIIth Report of the International Committee on Taxonomy of Viruses (Edited by: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA). New York: Elsevier Academic Press 2005, 43–47.
- Lavigne R, Seto D, Mahadevan P, Ackermann H-W, Kropinski AM: Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using BLASTP-based tools. Research in Microbiology 2008, 159:406–414.PubMedView Article
- Gadaleta P, Zorzopulos J:Kluyvera bacteriophage Kvp1: a new member of the Podoviridae family phylogenetically related to the coliphage T7. Virus Research 1997, 51:43–52.PubMedView Article
- Thomas JA, Hardies SC, Rolando M, Hayes SJ, Lieman K, Carroll CA, et al.: Complete genomic sequence and mass spectrometric analysis of highly diverse, atypical Bacillus thuringiensis phage 0305f8–36. Virology 2007, 368:405–421.PubMedView Article
- Zuber S, Ngom-Bru C, Barretto C, Bruttin A, Brussow H, Denou E: Genome analysis of phage JS98 defines a fourth major subgroup of T4-like phages in Escherichia coli . Journal of Bacteriology 2007, 189:8206–8214.PubMedView Article
- Walkinshaw MD, Taylor P, Sturrock SS, Atanasiu C, Berge T, Henderson RM, et al.: Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA. Molecular Cell 2002, 9:187–194.PubMedView Article
- Sturrock SS, Dryden DT, Atanasiu C, Dornan J, Bruce S, Cronshaw A, et al.: Crystallization and preliminary X-ray analysis of ocr, the product of gene 0.3 of bacteriophage T7. Acta Crystallographica Section D-Biological Crystallography 2001, 57:1652–1654.View Article
- Studier FW, Movva NR: SAMase gene of bacteriophage T3 is responsible for overcoming host restriction. J Virol 1976, 19:136–145.PubMed
- Marchand I, Nicholson AW, Dreyfus M: High-level autoenhanced expression of a single-copy gene in Escherichia coli: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements. Gene 2001, 262:231–238.PubMedView Article
- Robertson ES, Aggison LA, Nicholson AW: Phosphorylation of elongation factor G and ribosomal protein S6 in bacteriophage T7-infected Escherichia coli . Molecular Microbiology 1994, 11:1045–1057.PubMedView Article
- Condron BG, Atkins JF, Gesteland RF: Framshifting in gene 10 of bacteriophage T7. Journal of Bacteriology 1991, 173:6998–7003.PubMed
- Alam SL, Atkins JF, Gesteland RF: Programmed ribosomal frameshifting: much ado about knotting! Proceedings of the National Academy of Sciences of the United States of America 1999, 96:14177–14179.PubMedView Article
- Chandler M, Fayet O: Translational frameshifting in the control of transposition in bacteria. Molecular Microbiology 1993, 7:497–503.PubMedView Article
- Reeder J, Giegerich R: Design implementation and evaluation of a practical pseudoknots folding algorithm based upon thermodynamics. BMC Bioinformatics 2004, 5:104–115.PubMedView Article
- Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual Third Edition Cold Spring Harbor, New York: Cold Spring Harbor Press 2001.
- Laslett D, Canback B: ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Research 2004, 32:11–16.PubMedView Article
- Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Research 1997, 25:955–964.PubMedView Article
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 1997, 25:3389–4022.PubMedView Article
- Ermolaeva MD, Khalak HG, White O, Smith HO, Salzberg SL: Prediction of transcription terminators in bacterial genomes. Journal of Molecular Biology 2000, 301:27–33.PubMedView Article
- Lavigne R, Sun WD, Volckaert G: PHIRE, a deterministic approach to reveal regulatory elements in bacteriophage genomes. Bioinformatics 2004, 20:629–6135.PubMedView Article
- Darling AC, Mau B, Blattner FR, Perna NT: Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome 2004, 14:1394–1403.View Article
- Zafar N, Mazumder R, Seto D: CoreGenes: a computational tool for identifying and cataloging "core" genes in a set of small genomes. BMC Bioinformatics 2002, 3:12.PubMedView Article
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680–685.PubMedView Article
- Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res 2004, 14:1188–1190.PubMedView Article
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